USPTO Patents Application 10561839

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property Organization

International Bureau

(43) International Publication Date
11 January 2001 (11.01.2001)

PCT

(10) International Publication Number

WO 01/02539 Al

(51) International Patent Classification 7 :

A01K 67/027, C12N 5/02

(21) International Application Number: PCT/DK00/00308

(22) International Filing Date: 8 June 2000 (08.06.2000)

(25) Filing Language: English

(26) Publication Language: English

C12M 3/00, (81) Designated States (national): AE, AG, AL, AM, AT, AT
(utility model), AU, AZ, BA, BB, BG, BR, BY, CA, CH,
CN, CR, CU, CZ, CZ (utility model), DE, DE (utility
model), DK, DK (utility model), DM, DZ, EE, EE (utility
model), ES, FI, FI (utility model), GB, GD, GE, GH, GM,
HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KR (utility
model), KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG,
MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD,
SE, SG, SI, SK, SK (utility model), SL, TJ, TM, TR, TT,
TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.

(30) Priority Data:

PA 1999 00933

30 June 1999 (30.06. 1999) DK

(71) Applicant (for all designated States except US): THE
DANISH INSTITUTE OF AGRICULTURAL SCI-
ENCES [DK/DK]; P.O. Box 50, DK-8830 Tjele (DK).

(72) Inventor; and

(75) Inventor/Applicant (for US only): VAJTA, Gabor

[HU/AU]; 12/2 Kings Court, Clayton, VIC 3169 (AU).

(74) Agent: PATRADE A/S; Aaboulevarden 21, DK-8000
Aarhus C (DK).

(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG,
CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).

Published:

— With international search report.

For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.

OS

m

IT)

g (54) Title: MICROCHAMBER FOR EMBRYO CELL CULTURE

(57) Abstract: A process for in vitro culturing of cells, an apparatus for this process and applications of said apparatus and process
are provided. The cell culturing according to the invention is taking place in an especially formed receptacle (small wells formed
in within a larger well) which encourages culturing and growth encompassing development, fertilization and maturing, e.g. by its
^ small size (200-300 mm) and shape (tapered). Additionnally, a method for preparation of such a receptacle is disclosed. Finally,
applications of cell culturing within the disclosed receptacles are provided.

WO 01/02539

PCT/DKOO/00308

MICROCHAMBER FOR EMBRYO CELL CULTURE

The present invention relates to a process for in vitro culturing of cells, an apparatus
5 for this process and applications of said apparatus and process.

BACKGROUND OF THE INVENTION

The in vitro culturing of cells, whether prokaryotic or eukaryotic, is of obvious interest
10 when studying the growth, division and development of the cells per se in addition to
to products resulting hereof. Various instruments and remedies are available and
commercialised for cell culturing and usual devices for culturing comprise a dish or a
depression in a surface that is plane and smooth. A known type of this for those skilled
in the art is the so-called Petri dish. The culturing per se takes normally place within
15 said depression or dish and the cells are usually suspended in a nutrient fluid to
promote a cell development. The material used for production of these devices for
culturing is usually one or more polymeric compounds such as e.g. polystyrene.

As to the various types of cells to culture special interest has concentrated on
20 improvement of the in vitro culturing of eukaryotic cells including fertilisation hereof
and the culturing of the embryonic cells. A part of the background for this is the
wellknown interest in topics related to conditions for fertilisation in general. Therefore
there is an ongoing research within optimization of the conditions for fertilization
comprising both animals and humans. If the number of cells to culture is limited to a
25 small number, as is the case for human oocytes and for animal oocytes collected for
commercial purposes, the conditions for culturing are further challenged for what
reason additional efforts may be needed in order to achieve maximised conditions for
the culturing, including fertilization, growth and division of the cells. A number of
previous investigations have published that in vitro development of preimplantation
30 stage mammalian embryos seems to be facilitated by culturing the cells in groups, see
Vajta et al. 2000.

WO 01/02539 2 PCT/DK00/00308

In general by studying the prior art within conditions for cell culturing, it appears that
most researchers have published results mainly focused on various biochemical topics
such as e.g. optimisation of the media for culturing, the ratios of embryo/medium
volume applied and options for exchange and/or change of the nutrient medium during
5 the culturing. In contrast to this, considerations regarding the character of the physical
environment per se have remained relatively disregarded so far. Thus, also within the
most recent decade, very little attention has been devoted to improvements within the
topic of preparing an appropriate physical environment for the cell culturing to take
place. In more detail little research has been published as to the influence of the
10 physical characteristics of the room upon the culturing of cells.

The most commonly used method when culturing few embryos in order to decrease
the embryo/volume rates is to place the embryos in small droplets. By using this
approach, one problem is related to fluctuations of temperature and/or evaporation of

15 the liquid used. In order to minimize evaporation and temperature fluctuations an
appropriate amount of oil is commonly used as an overlay. However, this is often
accompanied with high diffusion of lipophilic material into the oil resulting in
decreased culturing conditions for the embryos. Another problem is the possible
accumulation of metabolic and toxic substances such as e.g. ammonia and oxygen-

20 derived free radicals. Also the presence of these catabolics gives rise to suboptimised
living conditions for the cells. A well known and frequently applied solution to this
has been to culture embryos in medium sized drops and perform changes of the
nutrient medium regularly, e.g. every 48 h. However, this manipulation has normally a
negative and harmful impact upon embryo development.

25

In continuation of this, new media and incubation systems have resulted in considerable
advances in the field of embryo production (Gardner et al., 1992; Takahashi and First,
1992; Gardner et al., 1994; Lane and Gardner; 1994; Trounson et aL, 1994; Vajta et al.
1997b; Holm et al., 1999). It appears from these sources that both an increase in the
30 developmental rates and much more stability in the ratios of development have been
obtained. Typically, the developmental rates about 5 years ago were reported to be

WO 01/02539 3 PCT7DKOO/00308

within the 5-15% range, while they are now in the 40-50% range (Callesen & Holm
1999).

SUMMARY OF THE INVENTION

5

As appears from the above, assessment in the public domain of the results of the cell
culturing experiments state that developmental rate including growth and division of
cultured cells is inter alia dependent upon the number of cells being assembled for
culturing. Cells being assembled should here be understood as cells being in mutual

10 contact, so that each cell is being in touch with at least one other cell. Hence, from the
prior art known to the inventors it appears that the developmental rate, comprising
growth rate and division rate of cells being cultured, increases when cells are
assembled for culturing. The inventors have found that cells assembled in a group of
cells seem to show an increase of the developmental rate within certain limits during

15 culturing subsequently to a rise of the number of cells belonging to said group.

It has surprisingly been shown by the present inventors, that the cell developmental
rate during culturing of a relatively little number of cells increases mutatis mutandis if
the cells are being cultured in wells, according to the invention. This observation is

20 surprising and provides an improvement of the culturing conditions, when only one
cell or a relatively small number of cells are to be cultured. Thus, by creating a
physical space around the cells being cultured, which space forms a micro
environment which is more narrow than reported in use hitherto, the cells in a
relatively small number grow and develop with an increased rate than previously

25 experienced with the size of well normally in use. Further details to this are given
below in addition to a disclosure of a methodology for the preparation of said wells.

DETAILED DESCRIPTION OF THE INVENTION

30 In a first aspect of the present invention the dependence of the physical environment
upon the in vitro growth of cells is featured. According to the invention cells are to be
understood as comprising prokaryotic as well as eukaryotic cells. By assessing

WO 01/02539

PCT/DK00/00308

published results from in vitro cell culturing experiments it appears that culturing of
cells assembled in groups of a relatively small number systematically differs in terms
of size of the found average developmental rate when compared to results based upon
groups of cells containing a bigger number of cells. Experiments have shown that
5 embodiments of the physical environment according to the invention especially
influences the rate of growth subsequently to the phase of maturation and of
fertilization.

It has surprisingly been shown by the present inventors, that the cell developmental
10 rate during culturing of a relatively little number of cells increases mutatis mutandis if
the cells are being cultured in wells, which are smaller than used previously. This
observation is surprising and provides an improvement compared to the prior art
within culturing conditions, when only one cell or a relatively small number of cells
are to be cultured. Thus, by creating a physical space around the cells being cultured,
15 which space forms a micro environment which is more narrow than reported in use
hitherto, the cells in a relatively small number grow and develop with an increased
rate than hitherto experienced with the size of well currently in use.

The current observation through the prior art is that the developmental rate of the cells
20 appear to be lowered when culturing a relatively small number of cells or even just
one cell. It has been surprisingly found as a first aspect of the present invention that
when culturing such a relatively small number of cells the in vitro rate of growth,
including processes such as fertilization, division of cells and maturing, is not lowered
if especially developed wells are used. Such a well, which is especially developed for
25 culturing of one cell or more cells forms according to the invention a well placed
within a larger surrounding well. A well according to the invention is termed a "well
of well", or a WOW.

The terminology of these two kinds of wells are as follows: A well present within a
30 bigger well is termed a well of a first kind and a well comprising a smaller well is
termed a well of a second kind, which hereinafter is used interchangeable with a
WOW (Well Of Well). A well as such is to be understood as a receptacle into a plane

WO 01/02539 5 PCTYDK00/00308

surface according to the present invention. This receptacle may be the result of a
mechanical process (manuable or machinable) before and/or after the manufacturing
of the pertinent material. The periphery of such a well may be of any form such as e.g.
circular, oval, rectangular and variations hereof. The depth should be so deep as to
5 allow space for the cells. The well should keep the cells within the well during the
practical operations performed.

Culturing and growth of a cell are used interchangeable and is to be understood as to
comprise all aspects and processes and results of processes during life cycles of cells
10 including eggs such as e.g. development, fertilization and maturing.

Furthermore, the present inventors have surprisingly found that the developmental rate
of the cells during the in vitro culturing of a relatively small number of cells increases
if the WOW is constructed in one way when culturing only one single cell and in a

15 different way when culturing more than one cell. According to the invention the
culturing of more than one cell should be performed with said cells assembled as a
group of cells. When culturing such a group of cells the WOW according to the
invention is larger in terms of periphery when culturing a group of cells than when
culturing only a single cell. A description of the two kinds of the WOW follows

20 below.

One characteristic of the appearance of the WOW for the in vitro culturing of a single
cell is that this WOW, seen vertically with the opening of the well being upwards, has
a cross-sectional area, that has a shape as the letter V or approximately as the letter V,
25 although the bottom per se has a form, which is more or less rounded. In a preferred
embodiment, the depth of this small WOW is 210-290 jum, preferably 225-275 \im
and particularly 240-260 |um. The inventors have found that an additional feature of
significance for the appropriate functionality of this small WOW is that the surface of
the inner wall of the WOW is smooth and without scratches.

30

The WOW for the in vitro culturing of cells assembled in a group has according to the
invention preferably a depth of approximately 260-340 |iim, preferably 275-325 fim

WO 01/02539 6 PCT/DK00/00308

and particularly 290-3 10 |j.m. The vertical cross-section of the inner shape of this large
WOW is rectangular or approximately rectangular and the bottom is flat. The whole
surface of the well is smooth and without scratches.

5 In a second aspect of the invention a process is disclosed for the preparation of a
WOW as a beneficial physical environment for the in vitro culturing of only one
single cell in addition to the preparation of a WOW for the culturing of at least two
cells assembled in a group.

10 A WOW according to the present invention and preferred for the in vitro culturing of a
single cell is prepared as follows. A rod is used, e.g. in the form of a needle or a
changeable tip for a soldering iron, made of a material, which is still solid at
temperatures used for the subsequent melting process, such as e.g. ground steel. The
rod surface may well have been subject to some kind of treatment leaving the surface

15 smooth, e.g. through covering with Teflon. Said material is heated, e.g. in a flame
preferably linked to a temperature controlling equipment, until the rod melts or gets
soft. Following the heating, the rod is pressed slightly until an appropriate receptacle
is formed into the surface of the bottom of this well, which beforehand is present in a
plane surface. The material in which the well is formed usually comprises one or more

20 polymeric compounds such as e.g. polystyrene. The melting point of this material is to
be lower than the melting point of the material of the rod. Upon touching said material
by the rod the material melts or softens and the rod makes a depression, i.e. a
receptacle into the bottom of the well of a second kind, thus making up a WOW. After
e.g. 15-20 seconds, the melted material solidifies and the rod is removed.

25 Approximately up to 15 WOWs may be made this way in each well of a second kind
and selected for this purpose. The WOWs and the wells of the second kind are then
cleaned by flushing by and/or incubating with one or more solvents suitable for
cleaning of the melted material such as e.g. an aqueous buffer, a serum/plasma
containing solvent or a nutrient fluid medium. The pertinent solvents may be used in

30 combination as one solvent or as single solvents.

WO 01/02539 7 PCT7DK00/00308

According to the invention a WOW preferred for culturing of at least two cells
assembled in a group is prepared as a WOW for culturing of a single cell except that
the rod used for making the depressions in the pertinent wells has a shape as a darning
needle with no needle point.

5

In the following, three examples (Examples 1, 2 and 3) of this are given for eggs from
cattle. Other types of cells could however also be used, e.g. eggs from other farm
animals such as pigs, from laboratory animals such as mice and from human beings.
Furthermore, the number of WOWs per well may well be lower compared to the
10 number of WOWs used for single cell culturing. This decrease is only due to practical
considerations. Approximately only up to 9 WOWs are made this way in each selected
well.

In the following, two examples (Examples 4 and 5) are given for the production of
15 different numbers of WOWs per well, and one example (Example 6) is given for the
influence of this on eggs from cattle.

EXAMPLES

20 EXAMPLE 1

Culturing of single embryonic bovine cells

Oocytes from the ovaries of slaughtered cows are subjected to in vitro insemination
according to conventional methods within the art (see e.g. Holm et al. 1999; Vajta et

25 al. 1997b). The developed embryos with several cells are separated into single cells
and are then cultured in wells (for comparison) and WOWs according to the invention.
The embryos are organised with one single cell per well and per small and per large
WOW, respectively. The cells are incubated the following 7 days in 400 jal medium
covered by 400 paraffine oil in four-well dishes (176740, Nunc, Life Technologies

30 AS, Roskilde, Denmark). The nutrient medium and the gaseous atmosphere in the well
and WOW and above the medium during the incubation of the cells is SOFaaci
(synthetic oviduct fluid medium supplemented with essential and non-essential amino

WO 01/02539 g PCT/DKOO/00308

acids, 0.34 mM tri-sodium citrate (Merck 1.06448, Darmstadt, Germany), 2.77 mM
myo-inositol (Sigma I 7508)) with 5% calf serum (CS; National Veterinary
Laboratory, Frederiksberg Denmark) and 5% C0 2? 5%0 2 90% N 2 with maximum
humidity, respectively. In a preferred embodiment the culturing takes place in a
5 Submarine Incubation System (Vajta et al. 1997a).

As a measure for the rate of development the blastocyst per oocyte ratios are
determined. The determination is performed using a stereomicroscope on day 7 and
the data are analyzed by logistic regression using a generalized linear model (SAS,
10 1993). Data differences between the incubations are compared by the contrast function
of the Genmod procedure (SAS, 1993).

Results are shown in Table 1 below in the first 3 rows. It appears that the average
developmental ratio is similar when the culture has been performed in wells or in
15 drops (32 % versus 34%), while it significantly increases when performed in WOWs
(60%).

Table 1 . Comparison of Day 7 blastocyst/oocyte ratios of one treatment group against
the control group. Different superscripts within the same module mean
20 significant difference (P<0.05). The four modules except for the control

module are referred to in the examples 1, 2 and 3.

Module

System

No. of Replicates

Blastocysts/oocytes j
(%)

Single zona intact
embryo
(Example 1)

Well

5

37 / 1 14 (32) a

Drop

5

42/ 125 (34) a !

WOW

6

102/ 168 (60) b

WO 01/02539 o PCT/DKOO/00308

Five zona intact embryos

Well

6

162/332 (49) a

1 (Example 2)

Drop

7

158/319 (50) a

WOW

9

213/347 (61) b

Single zona-digested
embryo

Well

4

17/ 69 (25)

(Example 3)

Drop

4

26/ 91 (29) a

WOW

8

136/256 (53) b

Control: 40-50 embryos

Well

24

973 /1775 (55)

EXAMPLE 2

Culturing of more at least two embryotic bovine cells assembled in a group

5 Oocytes from the ovaries of slaughtered cows are subjected to in vitro insemination
according to conventional methods within the art (see e.g. Holm et al. 1999; Vajta et
al. 1997b). The developed embryos are separated into groups of five embryotic cells
(=five embryos, each with several cells and are then cultured in wells (for comparison)
and WOWs according to the invention and organized with 5 cells per well and per

10 small and per large WOW, respectively. The cells are incubated the following 7 days
in 400 pi medium covered by 400 |ui paraffine oil in four-well dishes (176740, Nunc,
Life Technologies AS, Roskilde, Denmark). The nutrient medium and the gaseous
atmosphere in the well and WOW and above the medium during the incubation of the
cells is SOFaaci (synthetic oviduct fluid medium supplemented with essential and

15 non-essential amino acids, 0.34 mM tri-sodium citrate (Merck 1.06448, Darmstadt,
Germany), 2.77 mM myo-inositol (Sigma I 7508)) with 5% calf serum (CS; National
Veterinary Laboratory, Frederiksberg Denmark) and 5% C0 2 , 5%0 2 90% N 2 with

WO 01/02539

PCT/DKOO/00308

maximum humidity, respectively. In a preferred embodiment the culturing takes place
in a Submarine Incubation System (Vajta et al. 1997a).

As a measure for the rate of development the blastocyst per oocyte ratios are
5 determined. The determination is performed using a stereomicroscope on day 7 and
the data are analyzed by logistic regression using a generalized linear model (SAS,
1993). Data differences between the incubations are compared by the contrast function
of the Genmod procedure (SAS, 1993).

10 Table 1 shows the results as rows 4, 5 and 6, and it appears that the average
developmental ratio is similar whether the culturing has been performed in wells or in
drops (49% versus 50%), while it is significantly increased when performed in WOWs
(61%). Compared to single cultured embryos, an increase in developmental rate is
apparent from app. 33% to the app. 50% found in this experiment.

15

EXAMPLE 3

Culturing of single embryotic bovine cells without zona pellucida

Oocytes from the ovaries of slaughtered cows are subjected to in vitro insemination
20 according to conventional methods within the art (see e.g. Holm et al. 1999; Vajta et
al. 1997b). Removal of the zona pellucida is performed by an enzymatic reaction. A
preferred methodology for this proteolysis according to the invention is to incubate the
embryos for 2-3 min with 5 mg/ml Pronase (Sigma) according to Peura et al. (1998) in
Hepes-buffered TCM-199 medium at 35-37°C. Subsequently, the embryos are
25 incubated for 5 min in Hepes-buffered TCM -199 medium and 10% calf serum to
eliminate further pronase effect. The resulting zona free embryos are thereafter
separated into single embryos, each with several cells, and are then cultured in wells
(for comparison) and WOWs according to the invention. Thus, a single embryo is
cultured per well and per small WOW and per large WOW, respectively. The cells are
30 incubated the following 7 days in 400 jal medium covered by 400 j^l paraffine oil in
four-well dishes (176740, Nunc, Life Technologies AS, Roskilde, Denmark). The
nutrient medium and the gaseous atmosphere in the well and WOW and above the

WO 01/02539 I j PCT/DKOO/00308

medium during the incubation of the cells is SOFaaci (synthetic oviduct fluid medium
supplemented with essential and non-essential amino acids, 0.34 mM tri-sodium
citrate (Merck 1.06448, Darmstadt, Germany), 2.77 mM myo-inositol (Sigma I 7508))
with 5% calf serum (CS; National Veterinary Laboratory, Frederiksberg Denmark)
5 and 5% C0 2 , 5%0 2 90% N 2 with maximum humidity, respectively. In a preferred
embodiment the culturing takes place in a Submarine Incubation System (Vajta et aL,
1997a).

As a measure for the rate of development the blastocyst per oocyte ratios are
10 determined. The determination is performed using a stereomicroscope on day 7 and
the data are analyzed by logistic regression using a generalized linear model (SAS,
1993). Data differences between the incubations are compared by the contrast function
of the Genmod procedure (SAS, 1993).

15 Results are shown in table 1 and it appears that the average developmental ratio is
similar when the culture has been performed in wells compared to drops (app. 27%),
while it is significantly increased in WOWs (app. 53%).

EXAMPLE 4

20

Manufacture of single-cell WOWs

For the manufacture of single-cell WOWs a needle rod or a needle-like rod is used. An
ordinary sewing needle made of ground steel or its like may be used. The surface of
material chosen for the rod may include various compounds to facilitate the
25 manufacturing process. The surface may comprise compounds or constituents which
make the surface smooth and/or resistant to heat. An example of such a compound is
Teflon.

The chosen rod, e.g. a sewing needle made of appropriate ground steel, is heated in a
30 gas flame for 3-6 seconds or until the needle is hot enough to melt the material (e.g.
polystyrene) out of which the well is made. Following the heating the needle is
pressed slightly by hand into the bottom of a well in a four-well dish (176740, Nunc,

WO 01/02539 j2 PCT/DKOO/00308

Roskilde, Denmark). The well has an outer limit that is round-like and may form a
circle. Upon touching the polystyrene immediately melts and the needle makes a
depression into the bottom of the well. Such a depression is a receptacle for the cell
culturing and makes up a WOW. After normally 15-20 seconds, the melted material
5 solidifies and the needle is removed. Approximately up to 15 single cell WOWs are
made this way per well.

The wells are then filled with phosphate buffered saline (PBS) 4- 5% calf serum (CS;
National Veterinary Laboratory, Frederiksberg, Denmark) and rigorously flushed by
10 pipetting to remove air bubbles and possible loose, toxic material arised from the
melting process. PBS is subsequently replaced with SOFaaci (Holm et al. 1999) and
5% CS and incubates overnight at 39°C. After a second rigorous flushing of the wells,
the medium is replaced by a new aliquot of 500 ]il of SOFaaci and 5% CS, and
covered by 400 [i\ oil and used for the culturing.

15

EXAMPLE 5

Production of poly-cell WOWs

For the manufacture of poly-cell WOWs a needle rod or a needle-like rod is used. An
20 ordinary sewing needle made of ground steel or its like may be used. The surface of
material chosen for the rod may include various compounds to facilitate the
manufacturing process. The surface may comprise compounds or constituents which
make the surface smooth and/or resistant to heat. As an example the surface layer may
consist of or include Teflon.

25

The chosen rod, e.g. an ordinary sewing needle made of appropriate ground steel, is
heated in a gas flame for 3-6 seconds or until the needle is hot enough to melt the
material (e.g. polystyrene) of the well. Following the heating the needle is pressed
slightly by hand into the bottom of a well in a radiation sterilized dish delivered from
30 NUNC, Life Technologies AS, Roskilde, Denmark (production No. 176740: The well
has an outer limit which is round-like and may form a circle. Upon touching the
polystyrene immediately melts and a depression is made into the bottom of the well.

WO 01/02539 j 3 PCT/DKOO/00308

These depressions are receptacles for the cell culturing and make up the WOWs. After
normally 15-20 seconds, the melted material solidifies and the needle is removed.
Approximately up to 9 poly-cell WOWs are made this way per well.

5 The wells are then filled with phosphate buffered saline (PBS) + 5% calf serum (CS;
National Veterinary Laboratory, Frederiksberg, Denmark) and rigorously flushed by
pipetting to remove air bubbles and possible loose, toxic material arised from the
melting process. PBS is subsequently replaced with SOFaaci (Holm et al. 1999) and
5% CS and incubates overnight at 39°C. After a second rigorous flushing of the wells,
10 the medium is replaced by a new aliquot of 500 jal of SOFaaci and 5% CS, and
covered by 400 jlxI oil and used for the culturing.

EXAMPLE 6

15 Comparison of the developmental rates achieved in single and poly-cell WOWs

Using methods described in the previous examples 1, 2 and 3, embryos are cultured in
multiple versus single WOWs per well. The cells are incubated the following 7 days in
400 \il medium covered by 400 p.1 paraffine oil in four- well dishes (176740, Nunc,
Life Technologies AS, Roskilde, Denmark). The nutrient medium and the gaseous

20 atmosphere in the well and WOW and above the medium during the incubation of the
cells is SOFaaci (synthetic oviduct fluid medium supplemented with essential and
non-essential amino acids, 0.34 mM tri-sodium citrate (Merck 1.06448, Darmstadt,
Germany), 2.77 mM myo-inositol (Sigma I 7508)) with 5% calf serum (CS; National
Veterinary Laboratory, Frederiksberg Denmark) and 5% C0 2 , 5%0 2 90% N 2 with

25 maximum humidity, respectively. In a preferred embodiment the culturing takes place
in a Submarine Incubation System (Vajta et al., 1997a).

As a measure for the rate of development the blastocyst per oocyte ratios are
determined. The determination is performed using a stereomicro scope on day 7 and
30 the data are analyzed by logistic regression using a generalized linear model (SAS,
1993). Data differences between the incubations are compared by the contrast function
of the Genmod procedure (SAS, 1993).

WO 01/02539

14

PCT/DKOO/00308

Results are shown in Table 2 and it appears that the average developmental ratio is
similar when the culture has been performed in single compared to multiple WOWs
irrespective of the three types of cattle eggs tested.

Table 2. Comparison of Day 7 blastocyst/oocyte ratios in different modules using

multiple and single WOW per well.

Module

System

Replicate.
No.

Blastocysts/oocytes
(%)

Single zona intact
embryo

Multiple WOW

5

65/ 114 (57)

Single WOW

5

58/ 104 (56)

Five zona intact embryos

Multiple WOW

5

90/ 155 (61)

Single WOW

5

85/ 141 (60)

Single zona-digested
embryo

Multiple WOW

5

57/ 103 (55)

Single WOW

5

46/ 90 (51)

Differences within the same module are not significant (P>0.5).

WO 01/02539 j 5 PCT/DK00/00308

REFERENCES

CaUesen H, Holm P (1999) The results with Ovum-Pick-Up/In- Vitro Production
techniques. In: Ovum-Pick-Up and In- Vitro Production of Cow Eggs (in Danish).
Internal Report no. 125, Danish Institute of Agricultural Sciences, 6-15.

5

Gardner DK, Lane M, Spitzer A, Batt P (1994) Enhanced rates of cleavage and
development for sheep zygotes cultured to the blastocyst stage in vitro in the absence
of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups
stimulate development. Biology of Reproduction 50:390-400.

10

Gardner DK, Lane M, Spitzer A, Batt PA (1992) Amino acids and increased
embryo density stimulate development of sheep zygotes in vitro. Proceeding of
Australian Society of Reproductive Biology 24:90.

15 Holm P, Booth PJ, Schmidt MH, Greve T, CaUesen H (1999) High bovine
blastocyst development in a static in vitro production system using SOFaa medium
supplemented with sodium citrate and myo-inositol with or without serum.
Theriogenology 52:683-700.

20 Lane M, Gardner DK (1994) Amino acids increase mouse embryo viability.
Theriogenology 41 :233.

Peura TT, Lewis IM, Trounson AO (1998) The effect of recipient oocyte volume on
nuclear transfer in cattle. Molecular Reproduction and Development 50:185-191.

25

SAS Institute Inc., Cary, NC, USA (1993) Technical Report P-243, SAS/STAT
Software; The Genmod Procedure, Release 6.09.

WO 01/02539 ! 6 PCTYDKOO/00308

Takahashi Y, First NL (1992) In vitro development of bovine one-cell embryos;
influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 37:
963-972.

5 Trounson AO, Pushett D, Maclellan LJ, Lewis I, Gardner DK (1994) Current
status of IVM/IVF and embryo culture in humans and farm animals. Theriogenology
41:57-66.

Vajta G, Holm P, Greve T, Callesen H (1997a) The Submarine Incubation System,
10 a new tool for in vitro embryo culture. A technique report. Theriogenology 48:1379-
1385.

Vajta G, Holm P, Greve T, Callesen H (1997b) Survival and development of bovine
blastocysts produced in vitro after assisted hatching, vitrification and in-straw direct
15 rehydration. Journal of Reproduction and Fertility 1 1 1 :65-70.

Vajta G, Peura TT, Holm P, Paldi A, Greve T, Trounson AO, CaUesen H (2000)

New method for culture of zona-included or zona-free embryos: the Well Of the Well
(WOW) system. Molecular Reproduction and Development 55:256-264.

WO 01/02539

17

PCT/DKOO/00308

CLAIMS

1 . Process for in vitro culturing of one single cell or at least two cells assembled as a
group, characterised in that the culturing takes place in a well of a second

5 kind, which has a form of a receptacle, which is placed in the bottom of a well of a
first kind and in that the opening of the well of said second kind is smaller compared
to said well of said first kind.

2. Process for culturing of one single cell according to claim 1, wherein the cross-
10 sectional area of the well of said second kind has a shape like the letter V or

approximately like the letter V and wherein the surface hereof is smooth, plane and
without scratches.

3. Process for culturing of at least two cells according to claim 1, wherein the cross-
15 sectional area of the well of said second kind has a rectangular shape or approximately

a rectangular shape and wherein the surface hereof is smooth, plane and without
scratches.

4. Process for culturing of one single cell according to claim 2, wherein the depth of
20 the well of said second kind is 210-290 jum, preferably 225-275 \xm and in particular

240-260 |nm.

5. Process for culturing of at least two cells assembled as a group according to claim 3,
wherein the depth of the well of said second kind is 260-340 jam, preferably 275-325

25 \im and in particular 290-3 1 0 \xm.

6. Apparatus for culturing of one single cell or at least two cells assembled as a group,
comprising a smooth and plane material, wherein is made a well of a second kind,
being formed as a receptacle into said smooth and plane material, said well of said

30 second kind being characterised in that one or more wells of a first kind are
made into the bottom of said well of said second kind and in that the opening of the
well of said first kind is smaller compared to said well of said second kind.

WO 01/02539

PCT/DK00/00308

7. Apparatus for culturing of one single cell or at least two cells assembled as a group
according to any of the preceding claims characterised in that the wells of the
first and the second kind are present in a material comprising polymeric compounds.

5

8. Application of an apparatus according to claims 6-7 for cell culturing according to
claims 1-5, wherein the cultured cells are eukaryotic cells.

9. Application of an apparatus according to claims 6-8 for cell culturing according to
10 claims 1-5, wherein the cultured cells are taken from a group consisting of one single

embryotic bovine cell, at least two embryotic bovine cells, one single embryotic
bovine cell without zona pellucida, one single porcine embryo with zona pellucida and
at least two porcine embryos with zona pellucida.

15 10. Process for preparation of an apparatus according to claims 6-7, characteris
e d in that a hot rod is pressed slightly into the bottom of a well or receptacle present
in the surface of a material, which melts by touch of said rod.

INTERNATIONAL SEARCH REPORT

International application No.

PCT/DK 00/00308

A. CI ASSIFICATION OF SUBJECT MATTER

^J&^^^Jl^^^ ^cation ^ IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

IPC7: A01K, C12M, C12N

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic

data base consulted during the international search (name of data base and, where practicable, search terms used)

BOISIS. MEDLINE

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

WO 9220359 Al (MOUNT SINAI SCHOOL OF MEDICINE OF

THE CITY UNIVERSITY OF NEW YORK), 26 November 1992
(26.11.92), page 3, line 6 - line 19; page 4,
line 4 - page 5; page 7, line 25 - line 32,
and claims 4-13

1-9

US 5449620 A (JASPAR S. KHILLAN), 12 Sept 1995
(12.09.95), see col. 2 and claims

US 4894343 A (SHINJI TANAKA ET AL),
16 January 1990 (16.01.90)

10

1,2,4,5-9

1-9

P^j Further documents are listed in the continuation of Box C. See patent family annex.

* Special categories of cited documents:

"A" document defining the general state of the art which is not considered

to be of particular relevance
"E" erlier document but published on or after the international filing date
*L" document which may throw doubts on priority claimfs) or which is

cited to establish the publication date of another citation or other

special reason (as specified)
"O" document referring to an oral disclosure, use, exhibition or other

means

"P" document published prior to the international filing date but later than
the priority date claimed

later document published after the international filing date or priority
date and not in conflict with the application but cited to understand
the principle or theory underlying the invention

*X" document of particular relevance: the claimed invention cannot be
considered novel or cannot be considered to involve an inventive
step when the document is taken alone

"Y" document of particular relevance: the claimed invention cannot be
considered to involve an inventive step when the document is
combined with one or more other such documents, such combination
being obvious to a person skilled in the art

"8c" document member of the same patent family

Date of the actual completion of the international search

25 Sent 2000

Name and mailing address of the International Searching Authority

European Patent Office P.B 5818 Patentlaan 2

NL-2280 HV Rijswijk

Tel(+31 -70)340-2040, Tx 31 651 epo nl,

Fax(+31-70)340-3016

Date of mailing of the international search report

16. 10. Z000

Authorized officer

CARL-QLOF GUSTAFSSON/GH

Telephone No.

Form PCT /ISA/2 10 (second sheet) (July 1992)

INTERNATIONAL SEARCH REPORT

International application No.

PCT/DK 00/00308

C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category *

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

US 4975377 A (MARC E. KAY), 4 December 1990

(04.12.90), column 6, line 36 - line 59; column 9

WO 9713839 Al (PR0DUC0 AB), 17 April 1997

(17.04.97), page 10 - page 14, see figures and
claim 7

1,3,6-9

1,6-8

Form PCT/ISA/210 (continuation of second sheet) (July 1992)

INTERNATIONAL SEARCH REPORT

Information on patent family members

01/08/00

International application No.

PCT/DK 00/00308

Patent document
cited in search report

WO

9220359 A

Publication
date

Patent family
member(s)

Publication
date

26/11/92

AU
EP
JP
US
US
US

2016392 A
0585349 A
6507565 T
5512476 A
5627066 A
5691194 A

30/12/92
09/03/94
01/09/94
30/04/96
06/05/97
25/11/97

US

5449620

A

12/09/95

NONE

us

4894343

A

16/01/90

JP

2662215 B

08/10/97

JP

63129980 A

02/06/88

us

4975377

A

04/12/90

NONE

wo

9713839

Al

17/04/97

AT

192779 T

15/05/00

AU

708592 B

05/08/99

AU

6922596 A

30/04/97

CA

2234054 A

17/04/97

DE

59605198 D

00/00/00

EP

0853659 A,B

22/07/98

US

6037171 A

14/03/00

Form PCT/ISA/210 (patent family annex) (July 1992)